1. Field of the Invention
This invention relates to Epstein-Barr virus (EBV) associated disease, and specifically to the use of EBV specific peptides for diagnosis of EBV-associated disease.
2. Description of Related Art
Epstein-Barr virus (EBV) is a human herpesvirus which is endemic in all human populations. Most people are infected with the virus in early childhood and then carry the virus for life. If the initial infection is delayed until adolescence, infectious mononucleosis (IM) frequently results. IM is a self-limiting symptomatic disease with illness ranging from mild to severe. EBV is also linked with certain kinds of cancer. In the malarial belt of Africa, EBV is a contributory factor in the development of Burkitt""s lymphoma (BL) and in South-East Asia, the virus is linked to the high incidence of undifferentiated nasopharyngeal carcinomas (NPC). EBV has also been shown to be associated with certain malignancies occurring in organ transplant recipients and in some patients with acquired immune deficiency syndrome (AIDS).
Acute viral infection leads to the production of specific nuclear antigens (termed EBNA-I and EBNA-II), an xe2x80x9cearly antigenxe2x80x9d (EA) complex, viral capsid antigens (VCA), and other associated molecules. The xe2x80x9cearly antigen complexxe2x80x9d consists of the xe2x80x9cearly antigen-diffusexe2x80x9d (EA-D) and the xe2x80x9cearly antigen-restrictedxe2x80x9d (EA-R) antigens, based on their distribution in immunofluorescence assays. The antigens are distinguished by being localized in the cytoplasm plus nucleus (i.e. diffuse distribution) or in the cytoplasm only (i.e. restricted) and by their staining appearance in methanol-fixed cells. These EA antigens, with molecular weight 50-55 Kd, 17 Kd, and 85 Kd, respectively, are synthesized during the xe2x80x9clyticxe2x80x9d phase of EBV infection and not in transformed lymphoblastoid cells. Antibodies reactive with the early antigens are present during acute EBV infection and then disappear as the virus enters a phase of latency. The reappearance of anti-EA antibodies signals viral reactivation and provides some insight to the possible role of this virus in diseases such as nasopharyngeal carcinoma and Burkitt""s lymphoma.
Indirect evidence has suggested a possible role for EBV reactivation in patients with Sjogren""s syndrome, an autoimmune disorder characterized by lymphoid infiltrates of the salivary gland (the normal site for EBV latency). Since antibodies to EA antigens are detected by immunofluorescence assays, such antibodies cannot be detected in patients who possess antinuclear and anticytoplasmic antibodies as part of an autoimmune disease. Therefore it would be desirable to have an assay which uses purified EA molecules to allow measurement of anti-EA antibodies in patients with autoimmune diseases and to more accurately quantitate anti-EA antibodies in other patients with acute or reactivated EBV.
Recently, the DNA sequence of EBV was determined (Baer, et al., Nature 310:207, 1984) and the EA-D antigen localized in the genome. Using a monoclonal antibody directed against the EA-D protein, sufficient protein was purified to allow partial amino acid sequence determination and thus localization of the coding sequences. Using that information, it was possible to prepare a series of synthetic peptides based on the DNA sequence. The same strategy has proved useful in identifying immunologically important epitopes on the EBNA-I antigen (Rhodes, et al., J. Immunol., 134:211, 1985) and the EBNA-II antigens (Dillner, J. Proc. Natl. Acad. Sci. U.S.A., 81:4652, 1984) of EBV. A synthetic peptide derived from the EA-D molecule which contains an epitope reactive with immune human sera from patients with IM and other disease states has also been described (Fox, et al., J. Clin. Lab. Anal., 1:140, 1987).
Recent studies have shown that chemically synthesized polypeptides corresponding to short linear segments of a protein""s primary amino acid residue sequence can be used to induce antibodies that immunoreact with the native protein (Lerner, et al., Nature, 299:592, 1982; Sutcliffe, et al., Science, 219:260, 1983). In addition, some studies have shown that synthetic polypeptides can immunoreact with antibodies induced by native proteins (Rhodes, et al., J. Immunol., 134:211 1985). Thus, some synthetic polypeptides can immunologically mimic the immunogenic and antigenic determinants of native proteins.
Previous studies have examined the cellular immune response to EBV-induced antigens synthesized during the virus replication cycle (Pothen, et al., Int. J. Cancer, 49:656, 1991). The results demonstrated that some of the components of the early antigen (EA) complex were very effective in inducing a strong T-cell proliferative response similar to that previously noted with the major membrane glycoprotein, gp350/250 (Ulaeto, et al., Europ. J. Immunol., 18:1689, 1988). Both CD4+ and CD8+ lymphocyte populations from EBV-infected donors proliferated in the presence of polypeptides purified from the EA complex by immunoaffinity chromatography. The major polypeptide of EA-D and one of the major poly-peptides of EA-R were particularly effective in this T-cell recognition assay. The data suggested that these components of the EA complex might function as important target antigens in the immunosurveillance of EBV-infected or immortalized cells. Identification of the dominant T and B-cell epitopes expressed on EA-R complex polypeptides would provide information on the importance of the antibody responses to these components in the diagnosis and management of individuals with EBV-associated lymphoproliferative diseases.
The heterophile antibody test is currently the most widely used procedure for the diagnosis of acute IM. It is positive in 85-90% of adolescents and young adults, and a smaller amount of children with primary EBV infection. Specific EBV serology can be used to differentiate the heterophile-negative infections from mononucleosis-like illnesses caused by other agents including cytomegalovirus (CMV), human immunodeficiency virus (HIV), Toxoplasma gondii and adenovirus. Tests for individual measurements of antibodies to several different EBV-specific antigens, including the viral capsid antigen (VCA), EA-R, EA-D, and nuclear antigen (EBNA) have been described. In addition, differentiation of the IgG and IgM subclasses of the VCA can be helpful in diagnosis of EBV-associated disease. In heterophile negative serum, demonstration of the presence of VCA IgM and transient levels of early antigen antibodies may be considered diagnostic for acute IM.
About 80% of IM patients demonstrate a rise in antibodies to EA-D IgG during the acute phase of IM which may. then persist through the convalescent phase. EA-R antibodies appear transiently during the late convalescent phase, although young children and asymptomatic adults may exhibit an increase in antibodies during the acute phase. Early antigen IgG antibodies may persist for several months or even years following acute infection. Antibodies to EA are also seen in BL, NPC and in some cases of reactivated EBV infection. High levels of EA-R antibodies are generally seen only in the case of BL.
Although screening assays for detection of IM have been described, they do not identify as many as 10% of those patients with IM (U.S. Pat. No. 4,879,213, which utilizes K7B peptide). It would be desirable to develop improved methods to assay for the presence of EA-R or EA-D and anti-EA-R or anti-EA-D antibodies in a body sample so as to allow diagnosis of EBV involvement in disease, as well as diagnosis of the stage of disease, with greater sensitivity than already existing assays. It is also desirable to develop a means for distinguishing between EA IgG and IgM levels in order to identify acute versus convalescent phases of EBV-associated diseases, such as IM.
The present invention provides a simple and reliable diagnostic assay for detection of IgG and IgM antibodies to the diffuse (EA-D) and restricted (EA-R) components of the early antigen of the Epstein-Barr virus (EBV) in blood, and more specifically in serum. This novel assay can be used for diagnosis of EBV-associated disease; such as infectious mononucleosis (IM) for example, and can also be utilized to distinguish between individuals in the acute versus the convalescent phase of disease. The assay described herein is useful for detecting such EBV-associated diseases as IM with increased sensitivity over existing methods.